Project Summary: Cancer is a leading cause of death in the United States and worldwide. As such the President of the United States has recently established the White House Cancer Moonshot Task Force, the mission of which is to eliminate cancer as we know it. Part of this mission is to encourage development of novel cancer treatments. This innovative multi-PI proposal represents a novel approach to this challenge. Oncogenic activation of the RAS family of GTPases occurs in ~30% of cancers making it the most frequently mutated oncogene in human cancers. Despite a great deal of progress in our understanding of the biochemistry of RAS and it's role in tumorigenesis, development of effective therapeutic inhibitors of RAS to date has been disappointing. Thus, there remains a critical need to develop targeted inhibitors of this oncoprotein for treatment of patients with Ras-positive tumors. Using an unbiased, protein engineering approach, we have developed a highly specific and potent inhibitor of H-RAS and K-RAS based on the monobody platform. Monobodies are single-domain proteins of ~95 amino acids that achieve levels of affinity and selectivity similar to antibodies yet are insensitive to the redox potential of their environment. High affinity monobodies have been isolated to a diverse array of targets including the extracellular domain of receptors, kinases, steroid hormone receptors, and modular protein domains. Using this protein-based monobody inhibitor as a powerful experimental tool, we will probe the function of RAS in the tumorigenic process in ways that have not previously been possible. We propose three major aims to accomplish our goal of interrogating RAS function in oncogenesis. In Aim 1, we will use genetically encoded versions of our monobody inhibitor, termed NS1, to address unanswered questions regarding RAS function. In particular, NS1 blocks RAS through binding an allosteric interface important for dimerization of RAS and stimulation of signaling and transformation. Using NS1, we will address the importance of RAS dimerization in activation of multiple RAS effector pathways as well as probe the isoform specific difference in effector engagement. Aim 2 will employ a unique chemical-genetic approach to regulate NS1 expression in vivo to address whether targeting this novel allosteric interface interferes with RAS- dependent tumorigenesis. Building on our recent success with NS1, Aim 3 will develop isoform specific inhibitory monobodies to each RAS isoform and determine their mechanism of action. These studies represent a unique and powerful approach toward studying RAS and defining potential novel approaches to blocking RAS action. Thus, our work has the potential to make a major impact on cancer therapy. In addition, this project is highly relevant to the mission of National Cancer Institute's RAS Initiative at the Frederick National Laboratory for Cancer Research which is charged with targeting RAS-dependent cancers as well as President Obama's Cancer Moonshot initiative. We anticipate that our studies will answer important questions regarding RAS function in cancer while also providing powerful new experimental tools for the wider scientific community to utilize in interrogating RAS function.